Contribution of superoxide to reduced antioxidant activity of glycoxidative serum albumin

Hyperglycemia increases oxidative stress in various tissues and leads to diabetic cardiovascular complication. Dyslipidemia, such as an increase in oxidized low-density lipoprotein (LDL), is well recognized in diabetic patients with hyperglycemia. However, the mechanism by which hyperglycemia causes the increased LDL oxidation remains unclear. Albumin is the most abundant protein in the circulation, and can function as an antioxidant. Therefore, we examined whether glycoxidative modification inhibits the antioxidant activity of albumin to LDL oxidation and clarified the mechanism by which this modification may suppress its antioxidant activity. Human serum albumin (HSA) was incubated in phosphate-buffered saline with and without glucose at 37°C for up to 8 weeks under aerobic conditions (referred to as glycoxidation (goHSA) and oxidation (oHSA), respectively). Metal chelator-treated, nonoxidative HSA (chHSA) and freshly prepared HSA (fHSA) were used as controls. N ^ε-(carboxymethyl)lysine (CML), a glycoxidative product, was determined by enzyme-linked immunosorbent assay. Oxidation was estimated by measuring the thiols of the HSA molecule. Copper-mediated oxidation of LDL was conducted in the presence or absence of modified HSAs at 37°C for 6 days. Malondialdehyde and negative charge of LDL were measured. To clarify the mechanism of reduced antioxidant activity of HSA, we examined firstly the binding activity of modified HSAs to copper, and secondly the effects of free radical scavengers on the formation of malondialdehyde. CML was formed in goHSA in a time- and concentration-dependent manner. Both goHSA and oHSA significantly decreased the contents of free thiol groups compared to ch- and fHSAs. The antioxidant activity of goHSA to LDL oxidation was the lowest among various modified HSAs. The oHSA showed a moderate decrease in antioxidant activity. The binding activity of go- and oHSAs to copper was lower than that of ch- and fHSAs. The formation of MDA from LDL oxidation in the presence of goHSA was completely inhibited by Tiron (1,2-dihydroxy-3,5-benzenedisulfonic acid) and superoxide dismutase. In contrast, catalase and mannitol had no effect. Our results indicate that in vitro glycoxidation of HSA induced a marked loss of antioxidant activity of this molecule to copper-mediated oxidation of LDL, which may be caused by the generation of superoxide. Received: December 17, 2001 / Accepted: June 28, 2002 Acknowledgments The authors thank Drs. Ryoji Nagai and Seikoh Horiuchi (Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan) and Drs. Hiroyuki Itabe and Tatsuya Takano (Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan) for kindly supplying antibodies. We also thank Associate Professor Takeo Yamaguchi (Department of Chemistry, Faculty of Science, Fukuoka University) for the ESR experiment and Miss Satoko Nagano for her excellent technical assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 14570171) and in part by funds from the Central Research Institute of Fukuoka University (No. 016004). Correspondence to N. Sakata     

Successful embolization for uterine hemorrhage in a patient with acute promyelocytic leukemia

A successful embolization for life-threatening uterine hemorrhage is described in a patient with acute promyelocytic leukemia and disseminated intravascular coagulation. On admission the patient was in the 6th week of gestation with incomplete abortion and uterine hemorrhage. In addition to combination chemotherapy and anticoagulant therapy, dilatation and curettage were also performed. After the operation the uterine hemorrhage was progressively increased in amount and was not responsive to usual measures. Embolization of bilateral uterine arteries using Gelfoam was performed with a dramatic improvement of the hemorrhage.     

A case of pulmonary squamous cell carcinoma coexisting with pulmonary actinomycosis]

A 71-year-old man was referred to our hospital complaining of cough. Chest radiography revealed a mass opacity in the right upper lung field. A transbronchial biopsy specimen revealed non-specific inflammatory changes. Percutaneous lung aspiration biopsy under ultrasound guidance demonstrated gram-positive rods, suggesting actinomyces. On the diagnosis of pulmonary actinomycosis, the patient was treated with penicillin-G and his symptoms were relieved. In a three-month follow-up, the mass shadow in the right upper lung field was found to have increased in size. Squamous cell lung cancer was diagnosed on the basis of repeated transbronchial tumor biopsies, and right upper lobectomy was performed. Most cases of pulmonary actinomycosis have been diagnosed from post-surgical tumor specimens taken on suspicion of the presence of lung cancer. However, the lung cancer in this case was difficult to diagnose because the lung cancer was co-existent with pulmonary actinomycosis.     

Response of Human Insulinoma Cells to Extracellular Calcium Is Different from Normal B Cells

The preoperative determination of thelocalization of a small insulinoma is sometimesdifficult using routine imaging techniques. We have usedthe selective arterial calcium injection (SACI) test todetermine the location of the tumor preoperatively. Thepathophysiologic basis of the SACI test is based on theresponsiveness of insulinomas to calcium injected intothe feeding artery. In this study, we demonstrated the in vitro response of the insulinoma cellsto the extracellular calcium challenge by usingprimary-cultured insulinoma cells. Human insulinomacells were obtained from three patients. MIN6 cells(normal pancreatic B cells) were used as a control;their insulin response to various stimuli resembles thatof normal B cells. The insulin secretory dynamics inresponse to extracellular calcium were observed using a perfusion system. Second, the change ofthe concentration of cytosolic free calcium([Ca²⁺]_i) was monitored byfluorometry using fura-2/AM. When the concentration ofextracellular calcium ([Ca²⁺]_o) was changed from 2.54 mM to 10 mM, insulinsecretion from the insulinoma cells was markedlyincreased within 6 min (10- to 18-fold at maximum), andrapidly returned to the basal level; at the same time, [Ca²⁺]_i was immediatelyelevated and reached a peak within 1 min. In contrast,in the MIN6 cells, the insulin secretion and [Ca2+]iwere not significantly changed when[Ca²⁺]_o was switched to 10 mM. The results of these in vitro experiments agreedwith the clinical results of the SACI test. The positiveresponse of the insulinoma to the SACI test is probablydue to the different response of insulinoma cells to the extracellular calcium challengecompared with normal B cells. The role of[Ca²⁺]_i may be important in themechanism underlying the SACI test.     

Blockade of Death Ligand TRAIL Inhibits Renal Ischemia Reperfusion Injury

Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI). Many investigators have reported that cell death via apoptosis significantly contributed to the pathophysiology of renal IRI. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, and induces apoptosis and inflammation. However, the role of TRAIL in renal IRI is unclear. Here, we investigated whether TRAIL contributes to renal IRI and whether TRAIL blockade could attenuate renal IRI. AKI was induced by unilateral clamping of the renal pedicle for 60 min in male FVB/N mice. We found that the expression of TRAIL and its receptors were highly upregulated in renal tubular cells in renal IRI. Neutralizing anti-TRAIL antibody or its control IgG was given 24 hr before ischemia and a half-dose booster injection was administered into the peritoneal cavity immediately after reperfusion. We found that TRAIL blockade inhibited tubular apoptosis and reduced the accumulation of neutrophils and macrophages. Furthermore, TRAIL blockade attenuated renal fibrosis and atrophy after IRI. In conclusion, our study suggests that TRAIL is a critical pathogenic factor in renal IRI, and that TRAIL could be a new therapeutic target for the prevention of renal IRI.     

Lipoteichoic acid may affect the pathogenesis of PBC-like bile duct damage and might be involved in systemic multifocal epithelial inflammations in chronic colitis-harboring TCRα−/−×AIM−/− mice

Background: Chronic colitis-harboring TCRα^{? / ?} × AIM^{? / ?} mice showed PBC-like bile duct damage in the liver. Bacterial infection is one of the candidates for the pathogenesis of PBC. We demonstrated that the bacterial cell wall component lipotheicoic acid (LTA) was detected at sites of inflammation around damaged bile ducts in PBC patients. The aim of this study was to investigate the pathophysiology of the liver and other organs in TCRα^{? / ?} × AIM^{? / ?} mice.

Methods: Thirteen female TCRα^{? / ?} × AIM^{? / ?} mice were sacrificed at 24 weeks of age. The liver, stomach, small intestine, colon, pancreas, kidney and spleen were studied for pathological examination. Using anti-LTA antibody as the primary antibody, an immunohistochemical study was carried out.

Results: In the liver, LTA was mainly detected in the portal area with inflammation, and some of the cytoplasm of hepatocytes. Inflammations were also observed in the stomach, intestine, pancreas and kidney. Throughout the gastrointestinal tract, from the stomach to the colon, LTA was detected in the epithelium at sites of inflammation. Furthermore, LTA was detected around both pancreatic ducts with inflammation and distal renal tubules with inflammation.

Conclusions: The development of inflammations in the liver as well as extensive organs, strongly suggests a close relationship between bile duct damage and systemic multifocal epithelial inflammations, perhaps involving bacterial LTA, in TCRα^{? / ?} × AIM^{? / ?} mice.     

Evaluation of bioartificial renal tubule device prepared with human renal proximal tubular epithelial cells cultured in serum-free medium

Bioartificial renal tubule devices (BTD) use cell therapy to improve conditions commonly observed in recipients of artificial kidneys for treatment of kidney diseases. We previously reported significant improvement of the condition of acute kidney injury (AKI) animals after treatment with BTD prepared with lifespan-extended human renal proximal tubular cells (hRPTEC). However, a major obstacle to use of BTD for patients is their biological safety, because hRPTEC are cultured in medium containing fetal calf serum. To establish the biological safety of BTD, we prepared BTD with lifespan-extended hRPTEC cultured in a newly developed serum-free medium and compared these with BTD prepared with hRPTEC cultured in serum-containing conventional medium. Lifespan-extended hRPTEC cultured in serum-free medium (hRPTEC-SFM) can proliferate similar to hRPTEC cultured in serum-containing conventional medium (hRPTEC-CM). Comparison of leakage and of reabsorption of small molecules for BTD prepared with hRPTEC-SFM (BTD-SFM) with those for our previous BTD prepared with hRPTEC-CM (BTD-CM) showed transportation in these two types of BTD was almost identical. When AKI goats were treated with BTD-SFM for 26 h, increase of survival time and reduction of cytokine expression in blood cells were almost same as for AKI goats treated with BTD-CM. Quantification of the expression of some genes of hRPTEC in BTD revealed significant changes during BTD treatment for AKI goats. In conclusion, lifespan-extended hRPTEC-SFM work as well as hRPTEC-CM, and the biological safety of BTD for patients could be elevated without loss of function by preparation from hRPTEC-SFM.     

Does oral dryness influence pressure pain sensitivity in the oral mucosa of removable denture wearers?

Clinical Oral Investigations - This study aimed to determine if oral dryness is associated with oral pain sensitivity in removable denture wearers. The pressure pain threshold (PPT) in the mucosa...